The goal of the proposed research is to apply structural, biochemical, and biophysical analysis in an effort to discover novel inhibitors of HIV- reverse transcriptase ribonuclease H activity. HIV-1 reverse transcriptase (RT) is a central and characteristic enzyme of retroviruses, and is the target of many key anti-AIDS drugs. RT possesses two enzymatic activities, a DNA polymerase and a ribonuclease H (RNH). Although RT utilizes both DNA polymerase and RNH activities to carry out synthesis, to date, all RT inhibitors that have been approved for clinical use target the polymerase activity, not the RNH activity. Given the virus' ability to mutate and become drug resistant, it is crucial to explore RN inhibitors as potential anti-AIDS therapeutic agents. Though several RNH inhibitors have been found to inhibit RNH activity, visualization of inhibitor binding mechanisms could drive structure-based design of more effective antiviral drugs. The project goals are designed in order to provide training in the field of X-ray crystallography and utilize those skills in discovery and design of novel inhibitors of HIV-1 RT. The first goal will be to use X-ray crystallography to determine the structures of both active site and allosteric RNH inhibitors bound to HIV-1 RT and establish structure-activity relationships and detailed binding modes. In an effort toward the discovery of novel chemical classes of RNH inhibitors, a library of 1500 drug-like fragments will be analyzed for potential binding to HIV-1 RT using surface plasmon resonance and X-ray crystallography, and for RNH inhibition using a fluorescence based high throughput screen that looks specifically for RNA cleavage. The fragment screening approach allows for efficient search of chemical space and should lead to the identification of novel scaffolds for inhibiting HIV-1 RNH activity at both the active site and allosteric pockets.